1. Field of the Invention
The present invention relates to a substantially pure exopolysaccharide adhesin isolated from a particular strain of Staphylococcus epidermidis, to a general method capable of isolating this compound in substantially pure form, and to uses of said purified adhesin product as a vaccine for the production of antibodies effective against the binding of homologous bacterial cells to polymeric materials, and as a probe for the development of polymeric materials useful as catheters and medical prostheses.
2. Description of the Background Art
Both Staphylococcus aureus (coagulase-positive) and Staphylococcus epidermidis (coagulase-negative) have a characteristic propensity for invading skin and adjacent tissues at the site of prosthetic medical devices, including intravascular catheters, cerebrospinal fluid shunts, hemodialysis shunts, vascular grafts, and extended-wear contact lenses. Within 48 to 72 hours, relatively large numbers of staphylococci are demonstrable at the site of insertion of these foreign bodies. Archer, G. L., xe2x80x9cStaphylococcus epidermidis: The Organism, Its Diseases, and Treatment,xe2x80x9d in Remington, J. S., et al., eds., Current Clinical Topics in Infectious Diseases, McGraw-Hill, New York, 1986, pp. 25-46; Youmans, G. P., et al., The Biologic and Clinical Basis of Infectious Diseases, Saunders, Philadelphia, 1985, pp. 618-625, 738-9. It has been demonstrated that S. epidermidis cells attach and proliferate on the inner or outer surfaces of catheters, irrespective of their composition (polyethylene, polyvinylchloride, polyvinylfluoride, or polyester based materials).
Although the virulence of coagulase-negative staphylococci clearly is enhanced in the presence of a foreign body, the microbial factor(s) that permit these normal skin commensals to become noso-comial pathogens have not been well characterized. As adherence is believed to be the critical first step in the pathogenesis of coagulase-negative staphylococcal foreign-body infections, attention has focused on surface properties of these organisms that might mediate adherence to, and colonization of, polymeric prosthetic materials.
The most promising candidate for the source of a specific staphylococcal adhesin is an extracellular material often referred to as xe2x80x9cslime.xe2x80x9d It has been hypothesized that the slime substance may protect the S. epidermidis cells against antibiotics, as well as against natural host defense mechanisms. Youmans et al., supra; Peters, G., et al., Journal of Infectious Diseases 146:479-82 (1982).
It has been known since 1972 that coagulase-negative bacteria isolated from cerebrospinal fluid shunt infections elaborate a mucoid material that stains with alcian blue and is presumably a polysaccharide. Bayston, R., et al., Developmental and Medical Child Neurology, 14 (Supp. 27):25-8 (1972). The extracellular polysaccharide substance of slime-producing bacteria is a loose amorphous material composed of a range of low and high molecular weight polymers composed, in general, of neutral monosaccharides such as D-glucose, D-galactose, D-mannoso, L-fucoso, and L-rhamnose, and also contain amino sugars, uronic acid, and polyols such as ribitol and glycerol. Gristina, A. G., Science 237:1588-95 (1987). Glucose, galactose, phenylalanine, mannose, hexosamine, phosphorous, glycine and alanine have been found as components of the slime produced by S. epidermidis strains in clinical specimens unrelated to biomaterial infections. Ichiman, J., et al. J. Appl. Bact. 51:229 (1981). Isolates of such bacteria from sites of infections are more likely to produce slime than are random isolates from skin. Ishak, M. A., et al., Journal of Clinical Microbiology 22:1025-9 (1985). Moreover, slime-producing strains adhere well to a variety of polymeric materials. Christensen, G. D., et al., Infect. Immun. 37:318-26 (1982).
Coagulaso-positive staphylococci (S. aureus) are reported to produce multiple cell surface proteins which can be released from such cells by thermal extraction and which can be shown to bind to influenza virus-infected canine kidney cells. It was considered that S. aureus produces multiple cell surface protein adhesins. Sanford, B. A., et al., Infect. Immun. 52:671-5 (1986); Proc. Soc. Exp. Biol. Med. 181:104-11 (1986).
Identification of other microbial adhesins has been reported. Pier (U.S. Pat. No. 4,285,936, Aug. 25, 1981; U.S. Pat. No. 4,528,458, Mar. 25, 1986) discloses a method for partial purification of a polysaccharide antigen from Pseudomonas aeruginosa slime. Escherichia coli fimbrial protein adhesins have been identified and partially purified by several investigators (Orskov, I., et al., Infect. Immun. 47:191-200 (1985); Chanter, H., J. Gen. Microbiol. 125:225-243 (1983); Ferreiros, C. M., et al., Rev. espanol. de fisiolog, 39:45-50 (1983); and Moch, T., et al., Proc. Natl. Acad. Sci. 84:3462-6 (1987)).
Lectin-like glycoprotein adhesins have been identified in the Bacteroides fragilis group, and a 70 kDa adhesin has been purified by affinity chromatography (Rogemond, V., et al., Infect. Immun. 53:99-102 (1986)). Monoclonal antibody affinity chromatography was used to purify a 165 kDa surface protein of Mycoplasma pneumoniae which mediates attachment of such bacteria to target cells (Leigt, D. K., et al., J. Bacteriol. 157:678-80 (1984)), and to isolate a 150 kDa adhesin protein from Streptococcus sanguis FW213 (Elder, B. L., et al., Infect. Immun. 54:421-7 (1986)). A uroepithelial cell adhesin protein of 17.5 kDa was partially purified from fimbrii of Proteus mirabilis, a frequent cause of urinary tract infection (Wray, S. K., et al., Infect. Immun. 54:43-9 (1986)).
Ludwicka (Ludwicka, A., et al., Zbl. Bakt, Hyg. A 258:256-67 (1984)) fractionated by ion-exchange chromatography a phenol-saline extract of slime from S. epidermidis and obtained four crude fractions. Both the phenol-salite extract and two of the four crude fractions inhibited the attachment of bacterial cells to polymeric material. On the basis of the presence of monosaccharides in the fractions, the reaction of the fractions with lectins, and the complete inhibition of the production of the four fractions by pretreatment of the bacteria by tunicamycin (inhibitor of glycoprotein synthesis), the authors concluded that the extracallular slime substance is a complex of glycoconjugato (i.e., glycoprotein) character.
Hogt (Hogt, A. II., et al., Infect. Immun, 51:294 (1986) have also observed that crude extracellular products from the slime of homologous strains of S. epidermidis inhibit the adherence of homologous bacterial cells to polymeric materials used as catheters and prostheses. No information was provided in this report as to the chemical nature of the extracellular products.
Bacterial cells and materials derived from the surface of such cells have been used as vaccines to produce antibodies directed against homologous bacteria. Frank (Frank, R., et al., French Patent Application 85-07315, published Nov. 21, 1986) discloses a covalent conjugate between a capsular protein adhesin (MW-74 kDa) from Streptococcus mutans and a polysaccharide from the same (serotypically) organism, and the use of said conjugate as an anti-caries vaccine. Pier (Pier, G. B., et al., U.S. patents, supra) disclose a vaccine comprising a high molecular weight mucoid exopolysaccharide from Pseudomonas aeruginosa strain 2192 slime which induces in recipient animals an immunity to said organism. Sadowski (Sadowski, P., U.S. Pat. No. 4,443,549, Apr. 17, 1984; U.S. Pat. No. 4,652,498, Mar. 24, 1984; and EP 82401506.1, published Apr. 27, 1983) discloses monoclonal antibodies specific for surface adhesins of Escherichia coli and Pseudomonas aeruginosa which may be used for the therapeutic treatment of diseases induced by adhesin-bearing homologous bacteria in humans and animals. Nagy (Nagy, L. K., et al., Dev. Biol. Stand. 53:189-97 (1983)) discloses multi-adhesin vaccines for the protection of the neonatal piglet against Escherichia coli infections.
The inventors considered that, if a substantially pure capsular polysaccharide adhesin antigen could be isolated from the slime of strains of pathogenic S. epidermidis, a vaccine could be prepared from such an antigen that could be used to raise polyclonal antibodies in vivo in a human or animal, or monoclonal antibodies in hybridoma cells. Reasoning that adhesin-mediated colonization is required for the onset of pathogenesis, the inventors conceived that the polyclonal or monoclonal antibodies produced against the adhesin of the invention, by preventing the adherence of adhesin-bearing pathogenic bacteria to the recipient""s tissue cells or polymeric medical prostheses or catheters, represent a new means for preventing or treating diseases and infections due to S. epidermidis. 
Further, the substantially pure capsular polysaccharide adhesin of the invention is useful as a probe to test new polymeric materials for medical devices.
Therefore, in a preferred embodiment, the present invention provides a substantially pure polysaccharide from extracts of S. epidermidis RP-62 strain (an isolate from a patient with catheter-related bacteremia that produces copious quantities of slime) that mediates adherence to polymeric materials and also appears to be the capsule for this organism. In another preferred embodiment, the present invention provides a method for producing a substantially pure polysaccharide adhesin from extracts of S. epidermidis strain RP-62.
In another preferred embodiment, the substantially pure polysaccharide adhesin of the invention is used as a vaccine to raise in animals antibodies against said adhesin that inhibit the attachment of adhesin-bearing bacteria to polymeric materials.
The substantially pure polysaccharide of the invention may also be used as an antigen to produce monoclonal antibodies in hybridoma cells. Such monoclonal antibodies can be administered for prophylaxis or therapeutic purposes to humans or animals in order to prevent or reduce infections by coagulase-negative staphylococci.
In yet another preferred embodiment, the substantially pure polysaccharide adhesin of the invention is used to screen polymeric materials for resistance to attachment by bacteria.